A New 4α-Methylated Sterol from a Nephthea sp. (Nephtheidae) Bornean Soft Coral

Abstract

A new 4α-methyl sterol, 4α-methyl-ergosta-6,8(14),22E-triene-3β-ol (1), was isolated along with cholesterol from a Nephthea sp. Bornean soft coral. The structure of compound 1 was elucidated on the basis of spectroscopic analysis and comparison of the data with those of the related compounds.

Introduction

Marine organisms constitute a rich source of diverse and complex sterols; particularly among marine invertebrates the complexity of sterols arises through food chains and symbiotic relationships between organisms [1]. It has been observed that 4α-methyl steroids are often end products of steroids biosynthesis in the dinoflagellates and intermediates in steroids biosynthesis in animals and in other divisions of the Plant Kingdom [2,3]. Previous chemical investigations on soft coral have identified a variety of 4α-methyl sterols, possibly synthesized by the dinoflagellate symbiont of the soft coral [4,5,6,7,8,9]. The family Nephtheidae comprises many genera, among which Lemnalia, Paralemnalia, Capnella, Lithophyton, Dendronephthya, Scleronephthya, Stereonephthya and Nephthea have received considerable attention from organic chemists [10]. Among Octocorallia the genus Nephthea comprises a large variety of species. A literature search revealed that the genus Nephthea has afforded a variety of sesquiterpenes, diterpenes and steroids [4,5,6,11,12,13,14,15,16,17,18,19], but there have been no reports on chemical constituents of Malaysian soft corals. We have now examined an unidentified specimen collected from Sepanggar Island (Sabah, Malaysia), whose methanol extract afforded a new 4α-methyl sterol, identified as 4α-methyl-ergosta-6,8(14),22E-triene-3β-ol (1) and cholesterol (2). In this paper we report the isolation and structural determination by spectroscopic methods of new compound 1.

Figure 1. Structures of compounds 1 and 2.

Figure 1. Structures of compounds 1 and 2.

Results and Discussion

The sample was collected from Sepanggar Island (Sabah, Malaysia) and extracted with MeOH. The MeOH extract was concentrated and subsequently subjected to further purification to yield a new 4α-methyl sterol 1 and the known compound 2. Compound 2 was identified as cholesterol by comparing its spectral data with those reported in the literature [20].

Compound 1 was isolated as a white amorphous solid. HR-MS established a molecular formula of C₂₉H₄₆O, implying seven degrees of unsaturation. The ¹H-NMR spectrum of 1 clearly showed seven methyl signals at δ_H 0.65 (3H, s, H-19), δ_H 0.81 (3H, d, J = 6.9 Hz, H-27), δ_H 0.83 (3H, d, J = 6.9 Hz, H-26), δ_H 0.89 (3H, s, H-18), δ_H 0.91 (3H, d, J = 6.9 Hz, H-28), δ_H 1.02 (3H, d, J = 6.2 Hz, H-21) and δ_H 1.09 (3H, d, J = 6.2 Hz, H-29), four trisubstituted olefinic protons at δ_H 5.18 (1H, dd, J = 15.1, 8.3 Hz, H-22), δ_H 5.22 (1H, dd, J = 15.1, 6.9 Hz, H-23), δ_H 5.60 (1H, d, J = 10.3 Hz, H-7) and δ_H 6.15 (1H, dd, J = 10.3, 2.8 Hz, H-6), and one oxymethine proton at δ_H 3.15 (1H, m, H-3). The proton at δ_H 3.15 suggested the existence of the characteristic hydroxyl group at C-3 of 4α-methyl steroids [4]. The ¹³C-NMR and DEPT spectra of 1 also exhibited seven methyl carbons [δ_C 21.2 (q, C-21), 20.0 (q, C-26), 19.7 (q, C-27), 19.5 (q, C-18), 17.7 (q, C-28), 15.1 (q, C-29) and 12.4 (q, C-19),], six olefinic carbons [δ_C 147.3 (s, C-14), 135.5 (d, C-22), 132.2 (d, C-23), 126.2 (d, C-6), 126.0 (d, C-7) and 125.0 (s, C-8)] and one OH-bearing carbon (δ_C 77.3, d, C-3). The NMR and HRMS data could thus account for three of the seven degrees of unsaturation, suggesting the tetracyclic nature of 1.

All C–H correlations of 1 were detected in the HSQC experiment. The ¹H–¹H COSY spectrum exhibited partial structures a, b, c and d (Figure 2). Confirmation of the partial structures and their connectivity was made with the aid of the HMBC spectrum. HMBC correlations between H₃-19 and C-1/C-5/C-9/C-10 established partial structure a, could be connected to b through a quaternary carbon (C-10). HMBC correlations between H₃-18 and C-12/C-13/C-17 revealed partial structure b could be connected to c through a quaternary carbon (C-13). Furthermore, HMBC correlations between H₃-26/C-24, H₃-27/C-24 and H₃-28/C25 established the connection of partial structure c with d. Partial structures a and c were connected through fully substituted double bond between C-8 and C-14 by HMBC cross-peaks between H-7/C-8, H₂-15/C-8, H₂-15/C-14 and H₃-18/C-14. Based on available spectroscopic data obtained for this compound, there were no other available connection option then of C-8 to C-9. HMBC correlations from H₃-29 to C-3, C-4 and C-5 confirmed the existence of a 4α-methyl group. Based on these findings, the planar structure of 1 was concluded as shown in Figure 1.

Figure 2. ¹H-¹H COSY correlations (bold lines) and key HMBC correlations (H → C) of 1.

Figure 2. ¹H-¹H COSY correlations (bold lines) and key HMBC correlations (H → C) of 1.

The relative stereochemistry of compound 1 was deduced from the NOESY experiment as well as the coupling constants in the ¹H-NMR spectrum. The coupling constant between H-22 and H-23 (J = 15.1 Hz) suggested the double bond to have E configuration. Furthermore, as shown in Figure 3, the NOESY correlations observed between H-1α/H-9, H-2β/H₃-19, H-3/H-5, H-4/H₃-19, H-5/H-9, H-5/H₃-29, H-9/H-12α, H-11β/H₃-18, H-11β/H₃-19, H-12β/H₃-21 and H₃-18/H-20 revealed the relative configurations for each ring junction and chiral center. The configuration at C-24 was proposed by comparison of its NMR data with those of model compounds (Table 2) [21,22]. Thus, compound 1 was identified as 4α-methyl-ergosta-6,8(14),22E-triene-3β-ol.

Table 1. ¹H NMR and ¹³C NMR spectral data of compound 1 (recorded at 600/150 MHz in CDCl₃; δ in ppm, J in Hz).

Position	13C	1H (J in Hz)
1	35.1 (CH2)	1.69 (m, 1H)
		1.17 (m, 1H)
2	31.2 (CH2)	1.88 (m, 1H)
		1.54 (m, 1H)
3	77.3 (CH)	3.15 (m, 1H)
4	38.1 (CH)	1.38 (m, 1H)
5	51.2 (CH)	1.69 (dd, J = 9.6, 2.8 Hz, 1H)
6	126.2 (CH)	6.15 (dd, J = 10.3, 2.8 Hz, 1H)
7	126.0 (CH)	5.60 (d, J = 10.3 Hz, 1H)
8	125.0 (C)
9	48.5 (CH)	1.92 (m, 1H)
10	36.4 (C)
11	19.7 (CH2)	1.60 (m, 1H)
		1.45 (m, 1H)
12	36.8 (CH2)	1.98 (ddd, J = 12.4, 3.5, 3.5 Hz, 2H)
		1.27 (m, 1H)
13	43.5 (C)
14	147.3 (C)
15	25.0 (CH2)	2.35 (m, 1H)
		2.27 (m, 1H)
16	28.0 (CH2)	1.75 (m, 1H)
		1.40 (m, 1H)
17	56.1 (CH)	1.19 (m, 1H)
18	19.5 (CH3)	0.89 (s, 3H)
19	12.4 (CH3)	0.65 (s, 3H)
20	39.6 (CH)	2.09 (m, 1H)
21	21.2 (CH3)	1.02 (d, J = 6.2 Hz, 3H)
22	135.5 (CH)	5.18 (dd, J = 15.1, 8.3 Hz, 1H)
23	132.2 (CH)	5.22 (dd, J = 15.1, 6.9 Hz, 1H)
24	43.0 (CH)	1.85 (m, 1H)
25	33.2 (CH)	1.46 (m, 1H)
26	20.0 (CH3)	0.83 (d, J = 6.9 Hz, 3H)
27	19.7 (CH3)	0.81 (d, J = 6.9 Hz, 3H)
28	17.7 (CH3)	0.91 (d, J = 6.9 Hz, 3H)
29	15.1 (CH3)	1.09 (d, J = 6.2 Hz, 3H)

Table 1. ¹H NMR and ¹³C NMR spectral data of compound 1 (recorded at 600/150 MHz in CDCl₃; δ in ppm, J in Hz).

Position	13C	1H (J in Hz)
1	35.1 (CH2)	1.69 (m, 1H)
		1.17 (m, 1H)
2	31.2 (CH2)	1.88 (m, 1H)
		1.54 (m, 1H)
3	77.3 (CH)	3.15 (m, 1H)
4	38.1 (CH)	1.38 (m, 1H)
5	51.2 (CH)	1.69 (dd, J = 9.6, 2.8 Hz, 1H)
6	126.2 (CH)	6.15 (dd, J = 10.3, 2.8 Hz, 1H)
7	126.0 (CH)	5.60 (d, J = 10.3 Hz, 1H)
8	125.0 (C)
9	48.5 (CH)	1.92 (m, 1H)
10	36.4 (C)
11	19.7 (CH2)	1.60 (m, 1H)
		1.45 (m, 1H)
12	36.8 (CH2)	1.98 (ddd, J = 12.4, 3.5, 3.5 Hz, 2H)
		1.27 (m, 1H)
13	43.5 (C)
14	147.3 (C)
15	25.0 (CH2)	2.35 (m, 1H)
		2.27 (m, 1H)
16	28.0 (CH2)	1.75 (m, 1H)
		1.40 (m, 1H)
17	56.1 (CH)	1.19 (m, 1H)
18	19.5 (CH3)	0.89 (s, 3H)
19	12.4 (CH3)	0.65 (s, 3H)
20	39.6 (CH)	2.09 (m, 1H)
21	21.2 (CH3)	1.02 (d, J = 6.2 Hz, 3H)
22	135.5 (CH)	5.18 (dd, J = 15.1, 8.3 Hz, 1H)
23	132.2 (CH)	5.22 (dd, J = 15.1, 6.9 Hz, 1H)
24	43.0 (CH)	1.85 (m, 1H)
25	33.2 (CH)	1.46 (m, 1H)
26	20.0 (CH3)	0.83 (d, J = 6.9 Hz, 3H)
27	19.7 (CH3)	0.81 (d, J = 6.9 Hz, 3H)
28	17.7 (CH3)	0.91 (d, J = 6.9 Hz, 3H)
29	15.1 (CH3)	1.09 (d, J = 6.2 Hz, 3H)

Figure 3. Key NOESY correlations of 1.

Figure 3. Key NOESY correlations of 1.

Table 2. Partial ¹³C-NMR spectral data of the model compounds (crinosterol and brassicasterol) and 1.

Position	Crinosterol (24S)	Brassicasterol (24R)	Compound 1
	13C	13C	13C
24	43.12	42.90	42.95
25	33.28	33.16	33.19
26	19.69	20.02	20.04
27	20.19	19.69	19.73
28	18.08	17.68	17.71

Table 2. Partial ¹³C-NMR spectral data of the model compounds (crinosterol and brassicasterol) and 1.

Position	Crinosterol (24S)	Brassicasterol (24R)	Compound 1
	13C	13C	13C
24	43.12	42.90	42.95
25	33.28	33.16	33.19
26	19.69	20.02	20.04
27	20.19	19.69	19.73
28	18.08	17.68	17.71

Experimental

General

Optical rotations were measured on an AUTOPOL IV automatic polarimeter (Rudolph Research Analytical). ¹H-NMR (600 MHz) and ¹³C-NMR (150 MHz) spectra were recorded with a JEOL ECA 600, with TMS as internal standard. HR-ESI-TOFMS spectrum was obtained with LCMS-IT-TOF (Shimadzu) in ESI mode. HPLC was conducted on a Waters 600 using UV detector, Luna 5μ Phenyl-hexyl (10.0 × 250 mm) and Luna 5μ C18(2) 100A (10.0 × 250 mm). Preparative TLC was performed with silica gel plates (Merck, Kieselgel 60 F₂₅₄). Silica gel (Merck, Kieselgel 60, 70-230 mesh) was used for column chromatography. Analytical TLC was performed on Merck Kieselgel 60 F₂₅₄. Spots were visualized by UV light or by spraying with a 5% phosphomolybdic acid-ethanol solution.

Biological material

A specimen of Nephthea sp. was collected from Sepanggar Island, Sabah (6^o04’017’’N, 116^o04’836’’E), on January 24, 2008. The voucher specimen was deposited in the herbarium of Institute for Tropical Biology and Conservation, Universiti Malaysia Sabah (BORNEENSIS).

Extraction and isolation

The fresh soft coral (400 g wet wt) was extracted with MeOH at room temperature for 7 days. The crude extract was evaporated under reduced pressure and the residue was partitioned between EtOAc and H₂O. The EtOAc fraction was further partitioned with hexane and 90% MeOH. The hexane fraction (1.0 g) was fractionated by Si gel column chromatography with a step gradient of hexane and EtOAc in the ratio of 9:1, 8:2, 7:3, 1:1 and EtOAc. The fraction (237 mg) eluted with hexane/EtOAc (8:2) was further separated by a combination of preparative TLC with CHCl₃ and HPLC (Luna 5μ Phenyl-hexyl) with 80% MeCN to afford compound 1 (1.8 mg). The fraction (20 mg) eluted with hexane/EtOAc (7:3) was separated by repeated preparative TLC with CHCl₃ and hexane/EtOAc (3:1) to give compound 2 (3.2 mg).

Characterization of 4α-Methyl-ergosta-6,8(14),22E-triene-3β-ol (1)

White amorphous solid; [α]²⁵_D -36.9 (c 0.13, CHCl₃); HR-TOFMS m/z 411.3593 [M+H]⁺ (calcd. for C₂₉H₄₇O, 411.3621); ¹H-NMR and ¹³C-NMR spectral data: see Table 1.